Rotaviruses are the primary cause of severe dehydrating diarrhea in infants and young children, globally causing an estimated 2 million hospitalizations and 440,000 deaths per year in children under the age of 5. Due to its significant morbidity and mortality, an important goal of the Laboratory of Infectious Diseases remains the development of methods for controlling, preventing, and treating rotaviral disease. Accomplishing this goal would be helped by a more complete understanding of the molecular biology of rotavirus, notably those events in the viral life cycle connected to the replication of the virus's segmented double-stranded (ds)RNA genome. An expected outcome of this project is the development of a reverse genetics system that could be used in manipulating the genetic information of the virus. Such a system would provide an important tool for generating new and modifying existing rotavirus vaccines. This project may also lead to the identification of potential targets for antiviral components that can subvert the rotavirus replication cycle. Specifically, this project seeks to identify and characterize molecular signals in viral RNAs that function in the expression of rotavirus genes and in the packaging and replication of the rotavirus genome. The project also seeks to characterize the structure and function of viral proteins involved in these processes. These aims will be accomplished by a combination of procedures, which include (i) analysis of the replication and translation efficiencies of mutated viral mRNAs in cell-free systems, (ii) computer modeling and structural analysis (e.g., RNAse mapping, NMR spectroscopy) of the recognition signals in the mRNAs, (iii) characterization of the enzymatic and structural properties (e.g., analytical ultracentrifugation, X-ray crystallography, cryo-electron microscopy, CD spectroscopy) of recombinant viral proteins, and (iv) elucidation of the specificity and targets of the viral RNA-binding proteins by gel mobility shift assay and RNA-protein cross-linking. Studies performed in the last year have provided insight into (i) the atomic structure and/or enzymatic properties of NSP2 and NSP5, viral nonstructural proteins required for viroplasm formation and involved in genome replication and packaging, (ii) the atomic structure and enzymatic properties of the viral RNA-dependent RNA polymerase, VP1, including the role of the inner capsid protein, VP2, in the activation of the polymerase, and (iii) the location and function of cis-acting elements in viral RNAs important for polymerase recognition and for genome replication and packaging. These analyses have been particularly significant in understanding the protein-protein and protein-RNA interactions, and their connected activities, that drive the assembly of early replication intermediates in the viroplasms (viral factories) that form in the cytoplasm of rotavirus-infected cells.